ABSTRACT Our long-term goal is to understand the transcriptional regulation of interfollicular epidermal (IFE) differentiation. The IFE is maintained by proliferating basal layer stem cells that self-renew, but also divide asymmetrically to generate postmitotic progeny deposited into the suprababasal compartment. As these progeny cells move toward the skin's surface they form successively the spinous, granular and cornified layers. The distinct morphology of each epidermal layer, combined with matching sharp boundaries in the expression of landmark genes, has established a stepwise differentiation model for the IFE. We have focused on the later IFE differentiation stages and their control by Grhl3, an evolutionarily conserved transcriptional regulator of epidermal barrier formation. Grhl3 also promotes keratinocyte migration where it activates a gene expression program distinct from that in differentiation. In this renewal application, we propose to employ emerging single cell approaches to define in vivo transcriptional regulation of IFE differentiation and collective keratinocyte migration- -at a scale and resolution not heretofore possible. In Aim 1, we will re-define IFE differentiation based on single cell RNA-seq (scRNA-seq) analysis. Our recent scRNA-seq experiments suggest that many gene batteries with distinct functions have expression patterns that cross different IFE layers, and that there is a large population of transition cells between the basal layer and the first spinous layer. Our hypothesis is that rather than a stepwise process, IFE differentiation is better understood as a continuous process where every cell in the IFE is at a distinct differentiation stage. We will use a new hybridization-based single cell method, to match our scRNA-seq data with landmarks in the IFE. We will also use ATAC-seq, to correlate chromatin accessibility with single cell mRNA expression. In Aim 2, we will understand how Grhl3 and other IFE regulators act in vivo. Unexpectedly, Grhl3 loss leads to an accumulation of an abnormal IFE cell population with progenitor characteristics. We will test the hypotheses that in addition to its well described role in activating terminal differentiation genes, Grhl3 suppresses the formation of this abnormal progenitor cell population. In Aim 3, we will define cellular heterogeneity in the migrating epithelial wound front. We will test the hypotheses that different regions of the migrating wound front contain groups of keratinocytes which can be classified based on their transcriptome and chromatin state; that there are cell signals within and between different keratinocyte populations of the wound front; that Grhl3 regulates adhesion properties of follower cells; and that cell heterogeneity, cell-cell signaling, and role of Grhl3 change as wound healing progresses. These experiments are significant and innovative because they will be the first to comprehensively characterize in an unbiased way the in vivo transcriptome heterogeneity of the IFE in differentiation and migration--relevant to many skin diseases. The premise is strong, based on extensive literature, published work on the role of Grhl3, and our recent scRNA-seq data.